Method for producing chiral alpha-hydroxycarboxylic acids by enzymatic hydrolysis of chiral cyanohydrins

ABSTRACT

The invention relates to a method for producing chiral α-hydroxycarboxylic crystalline acids consisting in transforming cyanhydrins (R) or (S) into α-hydroxycarboxylic acids (R) or (S), respectively by enzymatic hydrolysis in the presence of  Rhodococcus erythropolis  NCIMB 11540.

CROSS REFERENCE TO RELATED APPLICATION

This application is a continuation-in-part of application Ser. No.10/544,103, filed Aug. 2, 2005, which is the US national phase ofinternational application PCT/EP2004/000859 filed 30 Jan. 2004 whichdesignated the U.S. and claims benefit of AT A285/2003, dated Feb. 27,2003, the entire content of which is hereby incorporated by reference inthis application

BACKGROUND AND SUMMARY OF THE INVENTION

Optically active α-hydroxycarboxylic acids are used, for example, asadditives to feeds, or in the production of pharmaceutical activecompounds, vitamins and liquid crystals.

These optically active α-hydroxycarboxylic acids may, in addition, beadvantageously converted, for example according to Effenberger et al.,Angew. Chem. 95 (1983) No. 1, page 50, into N-substituted opticallyactive α-amino acids which are otherwise prepared only with greatdifficulty.

Chiral α-hydroxycarboxylic acids are nowadays accessible chemically, byfermentation, or enzymatically.

The literature accordingly discloses a number of various methods forsynthesis of chiral α-hydroxy-carboxylic acids.

For instance, racemic cyanohydrins, with addition of suitablemicroorganisms, can be hydrolyzed to give the desired chiralα-hydroxycarboxylic acids.

Production of chiral α-hydroxycarboxylic acids, especially theproduction of optically active lactic acid or mandelic acid, fromracemic cyanohydrins using various microorganisms of the generaAlicaligenes, Pseudomonas, Acinetobacter, Rhodococcus, Candida etc. isdescribed, for example, in EP 0 449 684, EP 0 527 553, EP 0 610 048,etc.

From this prior art, it is also known that when a racemic cyanohydrin isenzymatically hydrolyzed to the conjugate α-hydroxycarboxylic acid inthe presence of a nitrilase, the problem occurs that the enzyme isinactivated within a short time and thus the desired α-hydroxycarboxylicacid is usually obtained only in low yields and concentrations. Thisalso applies to the use of nitrile hydratases, which convert thecyanohydrin to the conjugate α-hydroxyamide. The hydroxyamides can thenin turn be converted to the conjugate α-hydroxycarboxylic acids.

It is also known, for example from Angew. Chem. 1994, 106, page 1615f.,that optically active cyanohydrins may be hydrolyzed by concentratedhydrochloric acid, without racemization, to give the conjugate chiralα-hydroxycarboxylic acids. The optical purity of the chiralα-hydroxycarboxylic acids thus produced corresponds here to the opticalpurity of the chiral cyanohydrin used, even if this is obtained in situby enzyme-catalyzed addition of a cyanide group to a conjugate aldehydeor a ketone and is further processed without isolation or purification.

It is disadvantageous with this reaction that sensitive substrates aredecomposed, and the occurrence of corrosion.

It was an object of the present invention to find a method in whichnitriles as polar as chiral cyanohydrins can be converted using a mildand efficient method into the conjugate chiral hydroxycarboxylic acids,the hydroxycarboxylic acids having about the same enantiomeric purity asthe cyanohydrins.

Unexpectedly, this object has been achieved by the use of a suitableenzymatic activity of bacteria of the genus Rhodococcus.

The present invention therefore relates to a method for producing chiralα-hydroxycarboxylic acids, which comprises converting (R)- or(S)-cyanohydrins by enzymatic hydrolysis in the presence of at least onesuitable enzyme of a Rhodococcus species into the conjugate (R)- or(S)-α-hydroxycarboxylic acids.

BRIEF DESCRIPTION OF THE DRAWINGS

Reference will be made to the accompanying drawings wherein:

FIG. 1 is a plasmid map of pMS470Nhase7.3; and

FIG. 2 is a plasmid map of pMS470-33/3/1/11.

DETAILED DESCRIPTION OF THE INVENTION

In the inventive method, (R)- and (S)-cyanohydrins are converted into(R)- and (S)-α-hydroxycarboxylic acids with an optical purity of upto >99% ee.

(R)- and (S)-cyanohydrins which are produced by enzymatic or chemicallycatalyzed addition of a cyanide group to the corresponding aldehydes orketones serve as starting compounds.

Preferably the enzymatic addition of a cyanide group to thecorresponding aliphatic, aromatic or heteroatomic aldehydes or ketonesis catalyzed by a hydroxy nitrile lyase

Therefore, the present invention also relates to a method for producingchiral α-hydroxycarboxylic acids which comprises converting (R)- or(S)-cyanohydrins by enzymatic hydrolysis in the presence of a nitrilaseinto the conjugate (R)- or (S)-α-hydroxycarboxylic acids and wherein the(R)- or (S)-cyanohydrines are obtained by hydroxynitrile lyase catalyzedaddition of a cyanide group to the corresponding aliphatic, aromatic orheteroaromatic aldehydes or ketones.

A particularly suitable hydroxy nitrile lyase for use in thisapplication is a hydroxy nitrile lyase from Hevea braziliensis or fromPrunus anygdalis.

The enzymatic or chemically catalyzed addition of a cyanide group to thecorresponding aldehydes or ketones can be performed here in a similarmanner to the prior art, for example in a similar manner to EP 0 951561, EP 0 927 766, EP 0 632 130, EP 0 547 655, EP 0 326 063, etc.

Suitable starting compounds are the aldehydes and ketones cited in theprior art.

Examples of suitable aldehydes are aliphatic, aromatic or heteroaromaticaldehydes. Aliphatic aldehydes are taken to mean saturated orunsaturated aliphatic, straight-chain, branched or cyclic aldehydes.Preferred aliphatic aldehydes are straight-chain aldehydes having inparticular 2 to 18 carbon atoms, particularly preferably 2 to 12, whichare saturated or monounsaturated or polyunsaturated. The aldehyde canhave not only C—C double bonds, but also C—C triple bonds. The aldehydecan be unsubstituted or monosubstituted or polysubstituted by groupsinert under the reaction conditions, for example by optionallysubstituted aryl or heteroaryl groups, such as phenyl or indolyl groups,by C₁-C₆-alkyl, optionally substituted cycloalkyl groups, which can haveone or more heteroatoms from the group O, S, P or N, halogen, ether,alcohol, acyl, carboxylic acid, carboxylic ester, nitro or azido groups.

Examples of aromatic or heteroaromatic aldehydes are benzaldehyde orvariously substituted benzaldehydes, for instance 2-chlorobenzaldehyde,3,4-diflurobenzaldehyde, 4-methylbenzaldehyde, 3-phenoxy-benzaldehyde,4-fluoro-3-phenoxybenzaldehyde, in addition furfural,anthracene-9-carbaldehyde, furan-3-carbaldehyde, indole-3-carbaldehyde,napththalene-1-carbaldehyde, phthaldialdehyde, pyrazole-3-carbaldehyde,pyrrole-2-carbaldehyde, thiophene-2-carbaldehyde, isophthalaldehyde orpyridinealdehydes, etc.

Examples of ketones are aliphatic, aromatic or heteroaromatic ketones inwhich the carbonyl carbon atom is unevenly substituted. Aliphaticketones are taken to mean straight-chain, branched or cyclic ketones.The ketones can be saturated or monounsaturated or polyunsaturated. Theycan be unsubstituted or monosubstituted or polysubstituted by groupsinert under the reaction conditions, for example by optionallysubstituted aryl or heteroaryl groups such as phenyl or indolyl groups,by halogen, ether, alcohol, acyl, carboxylic acid, carboxylic ester,nitro or azido groups.

Examples of aromatic or heteroaromatic ketones are acetophenone, indolylacetone, etc.

Preference is given to (R)- or (S)-cyanohydrins of the formula

where R1 and R2 independently of one another are H, a C₁-C₆-alkyl orC₁-C₆-alkenyl radical which is optionally monosubstituted orpolysubstituted by substituents inert under the reaction conditions, ora phenyl radical which is optionally monosubstituted or polysubstitutedby substituents inert under the reaction conditions, with the provisothat R1 and R2 are not both H.

Preferred substituents inert under the reaction conditions are, forexample, halogens, such as fluorine, bromine and chlorine, C₁-C₆-alkylor C₁-C₆-alkoxy, ether, ester, acetals or optionally substituted phenyland phenyloxy.

Particularly preferably, suitable compounds for the inventive method are(R)- or (S)-cyanohydrins, for instance (R)- or(S)-2-hydroxy-4-phenylbutyronitrile, (R)- or (S)-2-chloromandelonitrile,(R)- or (S)-mandelonitrile, (R)- or (S)-4-methylmandelonitrile, (R)- or(S)-3-phenoxymandelonitrile, (R)- or(S)-2-hydroxy-2-methylheptanenitrile, (R)- or(S)-2-hydroxy-2-phenylpropionitrile, (R)- or(S)-2-hydroxy-3-pentenenitrile, (R)- or(S)-1-hydroxycyclohexane-nitrile, (R)- or (S)-acetophenonecyanohydrin.

The corresponding (R)- or (S)-cyanohydrin is then enzymaticallyhydrolyzed according to the invention.

The enzymatic hydrolysis is performed according to the invention in thepresence of at least one suitable enzyme of a Rhodococcus species.Rhodococcus species, unexpectedly, were found to have a nitrilasesand/or a nitrile hydratase/amidase enzyme system available which aresuitable to hydrolyze the nitrile function of nitriles which are polarin such a manner as the above-listed cyanohydrins.

Suitable Rhodococcus species where these enzymes can be derived from areselected from the group consisting of Rhodococcus erythropolis,Rhodococcus ruber and Rhodococcus rhodochrous

A preferred Rhodococcus species according to the invention isRhodococcus erythropolis NCIMB 11540

By means of the nitrile hydratase/amidase enzyme system of Rhodococcusspecies, the chiral cyanohydrins are hydrolyzed in the first step by thenitrile hydratase into the conjugate chiral hydroxyamide which is thenconverted in a second hydrolysis step by the amidase into thecorresponding chiral α-hydroxycarboxylic acid. The microorganism can beused in the inventive method in any desired form, for example in theform of ground cells, crude or purified enzymes, recombinant enzymes,immobilized cells or enzymes, lyophilized cells, or “resting cells”.

Preferably, use is made of recombinant enzymes, resting cells orlyophilized cells, particularly preferably recombinant enzymes orresting cells.

In addition to the direct use of nitrile hydratase/amidase-activepreparations of Rhodococcus species cells, the use of recombinantpreparations expressed in a suitable microorganism, for instance E.coli, Pichia pastoris, Saccharomyces, Asperagillus, K. Lactis, etc. is agood alternative. The corresponding genes are introduced using plasmidconstructs into suitable host cells, for example into E. Coli, Pichiapastoris, Saccharomyces, Asperagillus, K. Lactis host cells. By choosingan inducible promoter, not only the nitrile hydratase, but also theamidase, can be overexpressed in active form. In the case of amidase,far higher activity levels can be achieved than in the case ofcorresponding fermentation of the Rhodococcus cells.

The microorganism is then suspended in the desired form in an aqueousmedium, such as water or a buffer solution. Suitable buffer solutionsare, for example, phosphate buffer, for instance K/Na phosphate buffer,PBS buffer, butyrate buffer, citrate solutions, etc.

The pH of the buffer solution used should be in the range from pH 4.5 topH 11, preferably from 5.5 to 8.5.

The resultant suspension is then admixed with the corresponding chiralcyanohydrin. Since the chiral cyanohydrins are lipophilic compounds ofrestricted water solubility, the use of a solubilizer as cosolvent isnecessary to bring the cyanohydrins into solution in the aqueous medium.

Suitable solubilizers are, for example, organic solvents, surfactants,phase-transfer catalysts, etc.

Organic solvents which are suitable as cosolvent for the inventivemethod are those which firstly can dissolve the substrate sufficientlyand secondly have as little as possible adverse effect on the enzymeactivity.

Examples of these are dimethyl sulfoxide (DMSO), dimethylformamide(DMF), C₁-C₆-alcohols, for instance methanol, ethanol, isopropanol,1-butanol, 2-butanol, tert-butanol or 1-pentanol, toluene or tert-butylmethyl ether (TBME) or mixtures thereof.

Preferably, as cosolvent, use is made of DMSO, DMF, ethanol, isopropanolor mixtures thereof, and particularly preferably DMSO and DMF.

The cosolvent fraction should be between 0.5 and 20% by volume, based onthe total volume of the reaction solution.

Preferably, the cosolvent fraction is between 1 and 15% by volume, andparticularly preferably between 2 and 10% by volume.

The substrate concentration in the reaction solution should be in theinventive method in the range from 1 g/l to 100 g/l (based on the totalvolume of the reaction solution), the acceptance of a sufficiently highsubstrate concentration being the fundamental precondition for use ofthe inventive enzymatic hydrolysis on a preparative scale.

Preference is given to substrate concentrations up to 50 g/l,particularly preferably up to 25 g/l.

The substrate concentration which is possible to react depends on theenzyme quantity used. For efficient quantitative reaction, the firsthydrolysis step must proceed very rapidly in order to avoiddecomposition of the cyanohydrin and the resultant racemization, so thatrelatively high cell densities are required.

It is necessary to note in this case that sufficient mixing of thereaction system is ensured.

The cell quantity or enzyme quantity depends on the activity of themicroorganism in the form used, and also on the substrate concentrationand the cosolvent.

The pH of the reaction mixture should be between 4.5 and 11, preferablybetween 5.5 and 8.

If appropriate, in addition a suitable acid or acid salt, for instancephosphoric acid, boric acid, citric acid, etc. can be added to thereaction mixture to set the pH.

The inventive enzymatic hydrolysis is carried out at a temperature of 10to 60° C., preferably at 15 to 50° C., and particularly preferably at 20to 45° C.

After hydrolysis has been carried out to give the desired chiralα-hydroxycarboxylic acids, they are isolated from the reaction mixtureby means of a known technique, for instance centrifuging of the cells,extraction of the product after acidification by HCl (e.g.: pH 2) and ifappropriate further purification by activated carbon filtration andrecrystallization.

By means of the inventive use of suitable enzymes of Rhodococcusspecies, thus polar nitriles, such as chiral cyanohydrins, are convertedin a simple and efficient manner under mild conditions into theconjugate chiral α-hydroxycarboxylic acids, with no racemizationoccurring. The desired α-hydroxycarboxylic acids are obtained, dependingon the ee value of the cyanohydrin used, in a high optical purity of upto above 99% and at high yields of up to over 98%.

EXAMPLE 1 Production of the Biocatalyst

For the production of biomass of Rhodococcus erythropolis NCIMB 11540, acomplex standard medium (medium A, see Table 1) was used. The strainswere maintained on agar plates using medium A (solidification using 15g/l of agar). The plates were sealed by lateral wrapping with parafilmand stored in a refrigerator at 4° C.

Growth of the liquid cultures was performed in 1000 ml conical flaskshaving chicanes using 250 ml of medium A at 30° C. and 130 rpm.

Variant I (without preculture): About half of the biomass of an agarplate was suspended in 5 ml of sterile physiological common saltsolution. One cell suspension was added to 250 ml of culture medium.

Variant II (with preculture): For the preculture, some biomass of anagar plate was suspended in 5 ml of sterile physiological common saltsolution. One cell suspension was added to 100 ml of culture medium(=preculture). After growth for 20-24 h, 5 ml of this preculture wasadded to 250 ml of culture medium.

The cells were harvested by centrifugation at approximately 3000 rpm for30 min at 0-4° C. The cells were washed once with K/Na phosphate buffer(50 mM, pH 6.5). Then, the cells were resuspended in fresh buffer andeither lyophilized after shock freezing (reactions with lyophilizedcells, Example 2), or this cell suspension (approximately 6-8% of theculture volume) was used directly for the biocatalytic reactions(reactions with resting cells, Example 3). TABLE I Composition of mediumA Sterilization Concentration group Substance [g/l] I Na₂HPO₄ 4.97KH₂PO₄ 2.04 II MgSO₄.7H₂O 0.2 III CaCl₂.2H₂O 0.02 Ammonium iron(III)0.05 citrate Trace solution SL-6 1 ml/l IV Yeast extract 1 Meat peptone10 V Glucose 10

EXAMPLE 2 Reactions Using Lyophilized Cells on an Analytical Scale

31.6 mg, 52.6 mg and 105.2 mg of lyophilized cells were rehydrated in 10ml of phosphate buffer (50 mM, pH 6.5) for approximately 1 hour at 130rpm and 20-25° C. 475 μl aliquots of this cell suspension weretransferred to 1.5 ml Eppendorf reaction vessels and admixed with 25 μlof an approximately 200 mM substrate solution of2-hydroxy-4-phenylbutyronitrile in DMSO (3 mg, 5 mg and 10 mg ofcells/ml, substrate concentration approximately 10 mM, 5% DMSO). Thereaction was carried out in the Thermomixer at 30° C. and 1000 rpm.After 0, 2, 4, 6, 8, 10, 15, 20, 30, 60 and 120 minutes, in each caseone Eppendorf reaction vessel was admixed with 0.5 ml of 1N HCl. Aftercentrifugation (5 min, 13 000 rpm) and corresponding dilution, theconcentrations of cyanohydrin, hydroxyamide and hydroxy acid weredetermined by HPLC. TABLE 2 Hydrolysis of2-hydroxy-4-phenylbutyronitrile by lyophilized Rhodococcus erythropolisNCIMB 11540 cells (10 mg of cells/ml; substrate concentration: 10 mM)concentration (mM) of substrate, hydroxyamide and hydroxycarboxylic acidas a function of time (min) 0 min 10 min 20 min 30 min 60 min 100 min120 min Substrate 10 mM  1.9 Mm 0.7 mM 0.25 mM  0 mM   0 mM   0 mM Amide0 mM 4.7 mM 3.5 mM 3.2 mM 2 mM 1.1 mM 0.5 mM Acid 0 mM 3.3 mM 5.4 mM 6.1mM 7.5 mM   8.2 mM 8.5 mM

EXAMPLE 3 Enzymatic Hydrolysis Using Resting Cells and Lyophilized Cells

The biocatalyst was produced in a similar manner to Example 1, Variant1,2 culture flasks. After 20 hours (OD₅₄₆=3.5 and 1.8), cells werecentrifuged off from 4 times 10 ml aliquots of fermentation broth andwashed once with K/Na—PO₄ buffer (pH 6.5, 50 mM). Two of the cellsamples were lyophilized before activity determination, and the othertwo were used as resting cells.

The cells were resuspended in 1.8 ml K/Na—PO₄ buffer (pH 6.5, 50 mM)(lyophilized cells were shaken for 1 h for rehydration). The reactionwas started by adding 200 μl of a 200 mM substrate solution in DMSO(substrate concentration approximately 20 mM) and carried out at 30° C.and 130 rpm in the shaking cabinet. After 30 min, 60 min and 17 h, 200μl were withdrawn and admixed with 200 μl of 1N HCl. Aftercentrifugation (5 min, 13 000 rpm) and dilution, the conversion rateswere determined by means of HPLC. Only the substrate and the twoproducts were taken into consideration in this. The substrate used was(R)-2-chloromandelonitrile (ee >99%). The results are shown in Tab. 3.TABLE 3 Results of the reactions of culture 1 (OD₅₄₆ 3.5), substrate:(R)-2-chloromandelonitrile

   Resting cells Conver- Conver- sion   sion rate CH rate HA [%]¹   [%]²   Lyophilized cells      (19 mg/ml) Conversion Conversion rate CH  rateHA [%]¹   [%]² 30 min 100  2 40 <1 60 min —  4 41  0 17 h — 42 43 <1¹The conversion rate of cyanohydrin (CH) relates to both products(hydroxyamide and hydroxy acid).²The conversion rate of hydroxyamide (HA) is based on the amount ofhydroxy acid which was formed from the hydroxyamide present.

EXAMPLE 4 Enzymatic Hydrolysis Using Different Substrate Concentrations

Experiment 4.1:

In experiment 4.1, the reaction was carried out on an analytical scale(reaction volume 1 ml) using 3 substrate concentrations (2.2 g/l, 6.6g/l, 13.2 g/l).

The biocatalyst was produced according to Example 1, Variant II, 2 l offermentation medium, harvest after 20 hours (OD₅₄₆ 6.1). The cells from8 times 10 ml of fermentation solution were centrifuged off in culturetubes. The resultant cell mass was washed once in each case with 2 mlK/Na-phosphate buffer (pH 6.5, 50 mM). The contents of 2 tubes werelyophilized for determining the dry weight.

Weight: 1. 37 mg of lyophilized cells/10 ml fermentation solution

-   -   2. 30 mg

(This amount corresponded to approximately the use of cells/ml in thefollowing reactions.)

The contents of the remaining 6 tubes were resuspended in 950 μl ofbuffer (OD approximately 40) and transferred to Eppendorfs. To each ofthese cell suspensions were added 50 μl of variously concentratedsubstrate solutions (3 concentrations, parallel batches, 5% DMSO ascosolvent). The Eppendorfs were shaken on the Thermomixer at 30° C. and1000 rpm. For monitoring the conversion rate, in each case 200 μl werewithdrawn and admixed with 200 μl of 1N HCl. After centrifugation (5min, 13 000 rpm) and dilution, the conversion rates were determined byHPLC.

Following concentrations of (R)-2-chloromandelonitrile were used:

-   a. Substrate solution: 11 mg of (R)-2-chloro-mandelonitrile in 250    μl of DMSO (approximately 260 mM)    -   Substrate concentration in the batch: 2.2 g/l (13.1 mM)-   b. Substrate solution: 33 mg of (R)-2-chloro-mandelonitrile in 250    μl of DMSO (approximately 290 mM)    -   Substrate concentration in the batch: substrate concentration:        6.6 g/l (39.4 mM)-   c. Substrate solution: 66 mg of (R)-2-chloro-mandelonitrile in 250    μl of DMSO (approximately 1580 mM)    -   Substrate concentration in the batch: 13.2 g/l (78.8 mM)

The batches a-c are compared in Table 4.1 with reference to theformation of 2-chloromandelic acid (in %). In all batches, the hydroxyacid was formed quantitatively. It was found that even relatively highsubstrate concentrations are accepted without problems. TABLE 4.1Comparison of batches a-c with reference to the formation of(R)-2-chloromandelic acid in % 10 min 20 min 30 min 40 min 50 min a 77%86% 92% 94% 96% b 45% 70% 82% 89% 95% c 58% 85% 96% 98% 100% Experiment 4.2:

In experiment 4.2, the reaction was carried out using 2 differentsubstrate concentrations (10 g/l, 20 g/l) on a 5 ml scale.

The biocatalyst was produced according to Example 1, Variant II, 2 lfermentation medium, harvest after 19 hours (OD₅₄₆ 8.4). The cells wereresuspended in approximately 140 ml of buffer (resting cells, OD₅₄₆ 52).In each case 4.75 ml of this cell suspension were used for the enzymaticreactions.

Two different concentrations of (R)-2-chloro-mandelonitrile were studiedin parallel batches. The reaction was started by adding 250 μl ofsubstrate solution and was carried out in culture tubes in the shakingcabinet at 30° C. and 130 rpm. To monitor the conversion rate, in eachcase 200 μl were withdrawn and admixed with 200 μl of 1N HCl. Aftercentrifugation (5 min, 13 000 rpm) and dilution, the conversion rateswere determined by HPLC.

-   a. 50 mg of (R)-2-chloromandelonitrile, dissolved in 250 μl of DMSO    ([S]=60 mM, 10 g/l, cosolvent: 5% DMSO)

As soon as after 30 minutes, all of the cyanohydrin had reacted to formthe hydroxyamide, after 2 h, approximately 40% of hydroxy acid wereformed. After 20 h, the reaction was quantitative.

-   b. 100 mg of (R)-2-chloromandelonitrile, dissolved in 250 μl of DMSO    ([S]=120 mM, 20 g/l, cosolvent: 5% DMSO)

As soon as after 30 minutes, all of the cyanohydrin had reacted to formthe amide, after 18 h, 36% of hydroxy acid were formed. After 43 h, 41%of hydroxy acid were formed, thereafter no further reaction took place.

Experiment 4.3:

In experiment 4.3, reactions were carried out using 10 g/l and 15 g/l of(R)-2-chloromandelonitrile. In addition, after complete conversion, theee of the hydroxy acid formed was determined.

The biocatalyst was produced according to Example 1, Variant II, 2.75 lof the fermentation medium, harvest after 20 hours. The cells wereresuspended in approximately 200 ml of buffer (resting cells, OD₅₄₆ 44).In each case 4.85 ml of this cell suspension were used for the enzymaticreactions.

Two different concentrations of (R)-2-chloro-mandelonitrile were studiedin parallel batches. The reaction was started by adding 150 μl ofsubstrate solution and was carried out in culture tubes in the shakingcabinet at 40° C. and 150 rpm. The conversion rate was monitored bymeans of HPLC. At complete conversion, the hydroxy acid was extractedafter acidification and the ee was determined.

-   a. 50 mg of (R)-2-chloromandelonitrile, dissolved in 150 μl of DMSO    ([S]=60 mM, 10 g/l, cosolvent: 3% DMSO)

As soon as after 30 minutes, all of the cyanohydrin had reacted to formthe amide, after 2 h 80% of hydroxy acid were formed, after 19 h thehydrolysis to form the hydroxy acid was complete (product ee >99%).

-   b. 75 mg of (R)-2-chloromandelonitrile, dissolved in 150 μl of DMSO    ([S]=90 mM, 15 g/l, cosolvent: 3% DMSO)

As soon as after 30 minutes, all of the cyanohydrin had reacted to formthe amide, after 2 h, approximately 60% of hydroxy acid were formed,after 19 h the hydrolysis to form the hydroxy acid was complete (productee=99%).

EXAMPLE 5 Enzymatic Hydrolysis Using Different Cosolvents

Experiment 5.1:

In reactions on a 50 ml scale, DMSO was compared with EtOH as cosolvent.5% cosolvent were used, but the substrate concentration was only 4 g/l(R)-2-chloromandelonitrile.

Biomass from 8 times 250 ml (minus 80 ml, see Experiment 4.1) offermentation medium (OD approximately 6.1) was harvested after 20 h. Thecells were suspended in 100 ml of K/Na-phosphate buffer (pH 6.5, 50 mM).This cell suspension (OD 60) was used for the enzymatic reactions. Thereactions of (R)-2chloromandelonitrile, dissolved in DMSO or EtOH werecarried out in 100 ml ground glass joint conical flasks at 150 rpm and30° C. To monitor the conversion rate by means of HPLC, in each case 200μl of sample were admixed with 200 μl of 1N HCl, centrifuged (5 min, 13000 rpm) and diluted before measurement. After complete conversion, theee of the product was determined.

a. 50 ml of cell suspension were admixed with 200 mg of(R)-2-chloromandelonitrile (>99%) dissolved in 2300 μl of DMSO and 200μl of 0.1% H₃PO₄.

Substrate concentration: 4 g/l (24 mM), 5% DMSO as cosolvent

Product ee of (R)-2-chloromandelic acid: 97%

b. 50 ml of cell suspension are admixed with 200 mg of(R)-2-chloromandelonitrile (>99%) dissolved in 2300 μl of EtOH and 200μl of 0.1% H₃PO₄.

Substrate concentration: 4 g/l (24 mM), 5% EtOH as cosolvent

Product ee of (R)-2-chloromandelic acid: >99%

Experiment 5.2:

Here, the solvents DMSO, EtOH and ^(i)PrOH were again used at a fractionof 5%; the substrate concentration was 10 g/l of(R)-2-chloromandelonitrile. The reaction was carried out on a 5 mlscale.

Production of the biocatalyst Example 4, Experiment 4.2 (OD₅₄₆ 8.4). Ineach case 4.75 ml of the cell suspension (resting cells, OD₅₄₆ 52) wereused for the enzymatic reactions. Reactions of(R)-2-chloromandelonitrile (>99%) dissolved in DMSO, EtOH and i-PrOHwere carried out in culture tubes at 150 rpm and 30° C. (parallelbatches).

To monitor the conversion rate by means of HPLC, in each case 200 μl ofsample were admixed with 200 μl of 1N HCl, centrifuged (5 min, 13 000rpm) and diluted before measurement. After complete conversion, the eeof the product was determined.

a. 50 mg of (R)-2-chloromandelonitrile, dissolved in 250 μl of DMSO([S]=60 mM, 10 g/l, cosolvent: 5% DMSO)

As soon as after 30 minutes, all of the cyanohydrin had reacted to formthe hydroxyamide, after 2 h, approximately 40% of hydroxy acid wereformed. After 20 h the reaction was quantitative (product ee=95%).

b. 50 mg of (R)-2-chloromandelonitrile, dissolved in 250 μl of EtOH([S]=60 mM, 10 g/l, cosolvent: 5% EtOH)

As soon as after 30 minutes, all of the cyanohydrin had reacted to formthe amide, after 3 h, 35% of hydroxy acid were formed. After 19 h, thehydrolysis to form the hydroxy acid was 94% complete, and after 28 h,the reaction is virtually complete (product ee=97%).

c. 50 mg of (R)-2-chloromandelonitrile, dissolved in 250 μl of i-PrOH,([S]=60 mM, 10 g/l, cosolvent: 5% i-PrOH)

As soon as after 30 minutes, all of the cyanohydrin had reacted to formthe amide, after 3 h, 8% of hydroxy acid were formed. After 44 h, 64% ofhydroxy acid were formed (product ee=92.3).

EXAMPLE 6 Enzymatic Hydrolysis at Different Temperatures

Experiment 6.1:

In Experiment 6.1, the course of the reaction was compared at reactiontemperatures of 30° C., 35° C. and 40° C. The batches were carried outon a 5 ml scale using a substrate concentration of 10 g/l of(R)-2-chloromandelonitrile. After complete reaction, the ee of theproduct was determined.

The biocatalyst was produced according to Example 4, Experiment 4.2(OD₅₄₆ 8.4). In each case 4.75 ml of the cell suspension (resting cells,OD₅₄₆ 52) were used for the enzymatic reactions. Reactions of 50 mg of(R)-2-chloromandelonitrile (>99%) ([S]=60 mM, 10 g/l), dissolved in 250μl of DMSO (5%) were carried out at 3 different temperatures (30° C.,35° C., 40° C.) in culture tubes at 150 rpm (parallel batches).

To monitor the conversion rate by means of HPLC, in each case 200 μl ofsample were admixed with 200 μl of 1N HCl, centrifuged (5 min, 13 000rpm) and diluted before measurement. After complete reaction, the ee ofthe product was determined.

a. T=30° C.

As soon as after 30 minutes, all of the cyanohydrin had reacted to formthe amide, after 2 h, 42% of hydroxy acid were formed, afterapproximately 20 h the hydrolysis to form (R)-2-chloromandelic acid wascomplete (product ee=95%).

b. T=35° C.

As soon as after 30 minutes, all of the cyanohydrin had reacted to formthe amide, after 2 h, 65% of hydroxy acid were formed, after 19 h thehydrolysis to form (R)-2-chloromandelic acid was complete (productee=96.5%).

c. T=40° C.

As soon as after 30 minutes, all of the cyanohydrin had reacted to formthe amide, after 2 h, 86% of hydroxy acid were formed, after 19 h, thehydrolysis to form (R)-2-chloromandelic acid was complete (productee=97.9%).

Experiment 6.2:

In Experiment 6.2, the temperature was increased to 50° C.

The biocatalyst was produced according to Example 4, Experiment 4.3(OD₅₄₆ 44). In each case 4.85 ml of the cell suspension (resting cells,OD₅₄₆ 44) were used for the enzymatic reactions. Reactions of 50 mg of(R)-2-chloromandelonitrile (>99%) ([S]=60 mM, 10 g/l), dissolved in 150μl DMSO (3%) were carried out at 3 different temperatures (30° C., 40°C., 50° C.) in culture tubes at 150 rpm (parallel batches).

To monitor the conversion rate by means of HPLC, in each case 200 μl ofsample were admixed with 200 μl of 1N HCl, centrifuged (5 min, 13 000rpm) and diluted before measurement. After complete reaction, the ee ofthe product was determined.

a. T=30° C.

As soon as after 30 minutes, all of the cyanohydrin had reacted to formthe amide, after 2 h, 42% of hydroxy acid were formed, after 19 h, thehydrolysis to form the hydroxy acid was complete (product ee >99%).

b. T=40° C.

As soon as after 30 minutes, all of the cyanohydrin had reacted to formthe amide, after 2 h, 80% of hydroxy acid were formed, after 19 h thehydrolysis to form hydroxy acid was complete (product ee >99%).

c. T=50° C.

As soon as after 30 minutes, all of the cyanohydrin had reacted to formthe amide, after 2 h, 92% of hydroxy acid were formed, after 19 h, thehydrolysis to form hydroxy acid was complete (product ee >99%).

EXAMPLE 7 Reactions of Cyanohydrins of Aldehydes in the Presence ofRhodococcus erythropolis NCIMB 11540 on a Semipreparative Scale

For all reactions, K/Na-phosphate buffer (50 mM, pH 6.5) was used. Thereaction was followed by HPLC. After sampling, to stop the biocatalyticreaction, sample volumes were admixed with 1N HCl (parallel samples).After centrifugation (5 min, 13 000 rpm), the samples were diluted withHPLC eluent.

For workup, the biomass was centrifuged off for 30 min at 4° C. and 3000rpm and washed once with distilled H₂O. After acidifying the supernatantwith 1N HCl to pH 2, it was extracted 3-4 times with TBME.

Experiment 7.1:

The biocatalyst was produced according to Example 1, Variant II. 3 lfermentation medium, harvest after 20 hours (OD₅₄₆ 5.9). The cells wereresuspended in buffer to make approximately 180 ml (resting cells, OD₅₄₆80). 0.6 g (R)-2-chloromandelonitrile (ee >99%), dissolved in 1.5 ml ofDMSO was admixed with 60 ml of this cell suspension. The hydrolysis wascarried out at 30° C. and 150 rpm in the shaking cabinet. After 30 min,the cyanohydrin was completely hydrolyzed, after 17 hours, the reactionto give (R)-2-chloromandelic acid was complete.

Crude yield: 0.73 g (109%)

Product ee: >99%

Experiment 7.2:

In Experiment 7.2, some reaction parameters were varied. Standardconditions were 10 g/l of substrate and DMSO as cosolvent (here 2.5%). Asecond batch was carried out using 15 g/l of substrate, a further with10 g/l of substrate and DMF as cosolvent.

The biocatalyst was produced according to Example 1, Variant II. 3 l offermentation medium, harvest after 20 hours (OD₅₄₆ 6.8). The cells wereresuspended to give approximately 190 ml of buffer (resting cells, OD₅₄₆69). Three reactions were carried out.

Batch A: 0.3 g of (R)-2-chloromandelonitrile (ee >99%), dissolved in 750μl of DMSO were admixed with 30 ml of this cell suspension. Thehydrolysis was carried out at 40° C. and 150 rpm in the shaking cabinet.After 30 min, the cyanohydrin was completely hydrolyzed, after 5 hoursthe reaction to give (R)-2-chloromandelic acid was complete.

Crude yield: 0.31 g (93%)

Product ee: >99%

Batch B: 0.3 g of (R)-2-chloromandelonitrile (ee >99%), dissolved in 750μl of DMF were admixed with 30 ml of this cell suspension. Thehydrolysis was carried out at 40° C. and 150 rpm in the shaking cabinet.After 30 min, the cyanohydrin was completely hydrolyzed, after 5 hoursthe reaction to give (R)-2-chloromandelic acid was complete.

Crude yield: 0.30 g (90%)

Product ee: 98.5%

Batch C, 0.45 g of (R)-2-chloromandelonitrile (ee >99%), dissolved in750 μl of DMSO, were admixed with 30 ml of this cell suspension. Thehydrolysis was carried out at 40° C. and 150 rpm in the shaking cabinet.After 30 min, the cyanohydrin was completely hydrolyzed, after 5 hoursthe reaction to give (R)-2-chloromandelic acid was virtually complete.

Crude yield: 0.45 g (90%)

Product ee: >99%

Experiment 7.3:

Here, 2 batches having differing substrate concentration (batch A 10g/l, batch B 15 g/l) were carried out. Both reactions proceededvirtually at the identical speed and were complete after 2 hours.

The biocatalyst was produced according to Example 1, Variant II. 3 lfermentation medium, harvest after 20 hours (OD₅₄₆ 1.2). The cells wereresuspended in approximately 180 ml of buffer (resting cells, OD₅₄₆ 70).Two reactions were carried out.

Batch A: 0.8 g (R)-2-chloromandelonitrile (ee >99%), dissolved in 1.6 mlof DMSO, were added to the cell suspension (80 ml). The hydrolysis wascarried out at 50° C. and 150 rpm in the shaking cabinet. After 15 min,the cyanohydrin was completely hydrolyzed, after 2 hours, the reactionto give (R)-2-chloromandelic acid was complete.

Crude yield: 0.85 g (95%)

Product ee: >99%

Batch B: 1.2 g (R)-2-chloromandelonitrile (ee >99%), dissolved in 1.6 mlof DMSO, were added to the cell suspension (80 ml). The hydrolysis wascarried out at 50° C. and 150 rpm in the shaking cabinet. After 30 min,the cyanohydrin was completely hydrolyzed, after 2 hours, the reactionto give (R)-2-chloromandelic acid was complete.

Crude yield: 1.26 g (94%)

Product ee: 98.9%

Experiment 7.4:

The biocatalyst was produced according to Example 1, Variant II. 2.5 lof fermentation medium, harvest after 20 hours (OD₅₄₆ 6.9). The cellswere resuspended in approximately 160 ml of buffer (resting cells, OD₅₄₆63).

1.3 g of (R)-2-chloromandelonitrile (ee >99%), dissolved in 2.5 ml ofDMSO, were added to 140 ml of this cell suspension. The hydrolysis wascarried out at 40° C. and 150 rpm in the shaking cabinet. After 15 min,the cyanohydrin was completely hydrolyzed, after 3 hours the reaction togive (R)-2-chloromandelic acid was complete.

Crude yield: 1.43 g (98%)

Product ee: >99%

Experiment 7.5:

In this reaction, 1 g of mandelonitrile was hydrolyzed at a substrateconcentration of 8 g/l to give the corresponding hydroxy acid.

The biocatalyst was produced according to Example 1, Variant II. 2 l offermentation medium, harvest after 20 hours (OD₅₄₆ 8.4). The cells wereresuspended in approximately 120 ml of buffer (resting cells, OD₅₄₆ 74).The reaction, after deep-freezing of the biocatalyst, was carried outovernight.

1.0 g of (R)-(+)-mandelonitrile, dissolved in 2.4 ml of DMSO, was addedto the cell suspension (120 ml). The hydrolysis was carried out at 40°C. and 150 rpm in the shaking cabinet. After 15 min, the cyanohydrin wascompletely hydrolyzed, after 5 hours the reaction to give (R)-mandelicacid was complete.

Crude yield: 1.16 g (100%)

Product ee: 93%

EXAMPLE 8 Reactions of Cyanohydrins of Ketones in the Presence ofRhodococcus erythropolis NCIMB 11540 on a Semipreparative Scale

A K/Na phosphate buffer (50 mM, pH 6.5) was used. The reaction wasfollowed by TLC.

For the workup, the biomass was centrifuged off for 20 min at 4° C. and6000 rpm and washed once with distilled H₂O. After acidifying thesupernatant with 1N HCl to pH 2, it was extracted 3-4 times with TBME.

The biocatalyst was produced according to Example 1, Variant II. 2 l offermentation medium, harvest after 20 hours. The cells were resuspendedin approximately 60 ml of buffer (resting cells, OD₅₄₆ 60).

300 mg of (S)-acetophenone cyanohydrin (25% acetophenone, ee 94%),dissolved in 1 ml of DMSO, were added to the cell suspension. After 20h, the reaction was complete according to TLC and the product(containing 1-phenylethanol and traces of other impurities) wasextracted. The reaction proceeded without loss of enantiomeric purity.

Crude yield: 357 mg

EXAMPLE 9 Generation of Enzyme Preparations of Nitrile Hydratase andAmidase for Hydrolysis of Substituted Cyanohydrins by Means ofRecombinant Expression in E. coli

For expression of the nitrile hydratase, and also for the expression ofthe amidase, the pMS470 plasmid system was used. In addition to thereplicated elements, this plasmid has a selectable ampicillin resistanceand the Lac repressor gene lacI via an inducible tac promoter, whichpermits controlled overexpression of the cloned open reading frame.

Expression Plasmid for the Rhodococcus erythropolis NCIMB 11540 NitriteHydratase:

The plasmid map may be seen in FIG. 1. The plasmid bears the namepMS470Nhse7.3. In addition to the two gene sections of the nitrilehydratase (α- and β-subunit) it also contains a third open reading framewhich codes for an activator protein.

Expression Plasmid for the Rhodococcus erythropolis NCIMB 11540 Amidase:

Other than the case with nitrile hydratase, for the expression of theamidase of Rhodococcus erythropolis NCIMB 11540 only a single readingframe is necessary, and this was cloned in plasmid pMS470-33/3/1/11downstream of the tac promoter. FIG. 2 shows the plasmid map of thisconstruct.

Fermentation of the Recombinant Nitrite Hydratase and Amidase

Fermentation of the two enzymes was always performed by the generalprotocol developed for overexpression of enzymes in the pMS470 system.

Here, samples were:

inoculated from an overnight culture (ONC) into a main culturecontaining LB medium and antibiotic, in the shaking flask.

allowed to grow to the exponential phase

at OD₆₀₀ (optical density at 600 nm) 0.8 to 1.5, induced with IPTG(isopropylthiogalactopyranoside)

further induced for 18 h (protein expression)

harvested (centrifugation) and disintegrated (ultrasound)

Expression of the Nitrile Hydratase

The E. coli B BL21 cells transformed by pMS470Nhase7.3 (orpMSNhasetactac7.3) were isolated on LB-ampicillin plates and an ONC of100 ml of LB-ampicillin medium was inoculated with an individual colony.On the next morning a main culture consisting of 250 ml of LB-ampicillinmedium was inoculated in a 1000 ml chicane flask to an OD₆₀₀ of 0.01 to0.03 (Beckmann Photometer). The growth temperature was controlled at 25°C., since at higher temperatures insoluble inclusion bodies are formedexclusively. After the induction density was reached (OD₆₀₀=1 BeckmannPhotometer), the cultures were induced by adding IPTG to give aconcentration of 0.1 mM. In addition, the media were supplemented with0.1 mM ammonium iron(III) citrate. After reaching an OD₆₀₀>4, thecultures were harvested (centrifugation at approximately 3000-g for 15min) and washed once with approximately 100 ml of PBS buffer. The cellpellet was then resuspended in PBS buffer (approximately 5 ml totalvolume) and disintegrated using a ultrasonic probe (BRANSON Sonifier250, 60% power setting, constant sonication; 5 times 30 s each time witha 1 min pause for cooling) (visual control of completeness under themicroscope). The resultant crude lysates had a typical activity ofapproximately 100-250 U/ml (approximately 350-500 U/ml forpMSNhasetactac7.3), analyzed using methacrylonitrile as substrate underthe conditions listed hereinafter.

For preservation, the lysates were stored at −20° C. Storage at roomtemperature is associated with rapid loss of activity.

Activity determination: crude lysates were diluted 1:10 with PBS bufferimmediately before activity determination. 1.4 ml of a 40 mMmethacrylonitrile solution in PBS buffer were admixed with 20 μl of thediluted lysate and incubated at 28° C. (Eppendorf Thermomixer 5436). Atthe time point 0, 1, 2, 5, 10 and 15 minutes, 200 μl samples werewithdrawn and immediately the enzyme reaction was stopped in thesesamples by 800 μl of 0.17% phosphoric acid. After centrifugation (16000·g, 10 minutes), the samples were analyzed spectrophotometrically at224 nm (Perkin Elmer UV/VIS Spectrometer Lambda Bio). The increase inextinction was correlated with the increase in concentration ofmethacrylamide, an ε value of 0.57 l·mmol⁻¹·cm⁻¹ being used as areference.

Expression of Amidase

The E. coli B BL21 cells transformed by pMS470-33/3/1/11 were isolatedon LB-ampicillin plates and an ONC of 100 ml of LB-ampicillin medium wasinoculated with an individual colony. On the next morning a main cultureconsisting of 250 ml of SOC-ampicillin medium was inoculated into a 1000ml chicane flask to an OD₆₀₀ of 0.01 to 0.03 (Beckmann Photometer). Thegrowth temperature was controlled to 30° C., since fermentation at 37°C. leads exclusively to the formation of insoluble and inactive protein.After reaching the induction density (OD₆₀₀=1 Beckmann Photometer), thecultures were induced by adding IPTG to a concentration of 0.3 mM. Afteran induction time of 16 h, the cells were harvested (centrifugation3000·g, 10 min) and washed with sodium phosphate buffer (0.1 M, pH=7).The pellet produced was resuspended to approximately 5 ml total volumein wash buffer and disintegrated using an ultrasound probe (BRANSONSonifier 250, 60% power setting, constant sonication; 5 times 30 s eachwith 1 min pause for cooling) with constant cooling to completeness(visual control of completeness under the microscope). The crude lysatesthus produced were frozen for preservation at −20° C. The lysates had anactivity of approximately 75 U/ml, determined using acetamide (40 mM) assubstrate in PBS buffer at 37° C. (determination of released ammonium bythe indophenolblue method).

Determination of amidase activity:

The following solutions were used:

Substrate solution: 40 mM acetamide in PBS

Solution A: 10% (w/v) phenol in ethanol (95%)

Solution B: 0.5% (w/v) nitroprusside sodium in ddH₂O

Solution C: 100 g of trisodium citrate and 5 g of sodium hydroxide in550 ml of water

Solution D: 600 ml of commercially conventional sodium hypochloritesolution diluted to 1000 ml

Ammonium standards: 0, 80, 120, 200, 280, 400 μg/l of ammonium sulfatein water

1.4 ml of substrate solution were incubated at 30° C. (EppendorfThermomixer 5436) with 10 μl of enzyme dilution (1:10 in PBS). Theenzyme reaction was stopped in 100 μl samples after 0, 1, 2, 5, 10 and15 minutes using 20 μl of solution A. After withdrawal of the lastsample, the resultant solutions were diluted with 400 μl of water. Forcalibration, in addition, ammonium standard solutions (each 500 ml) wereadmixed with 20 μl of solution A. To samples and also to standards,thereupon 20 μl of solution B and 50 μl of a mixture of 4 parts ofsolution C with 1 part of solution D were added by pipette. Good mixingwas ensured by vortexing. The resultant samples and standards were stoodat 37° C. for 15 min. The resultant blue coloration, after dilution ofall samples and standards 1:10) with water, was quantified in thespectrophotometer (Perkin Elmer UV/VIS Spectrometer Lambda Bio) at 640nm. By correlating the increase in blue coloration over time with theextinction values of the standards, the activity (μmol of releasedammonium per minute) could be back-calculated.

EXAMPLE 10 Hydrolysis Using the Recombinant Enzyme

In these reactions, cloned nitrile hydratase of Rhodococcus erythropolisNCIMB 11540 was used as crude lysate of E. coli clone 7.3 (producedaccording to Example 9).

Procedure:

50 μl of crude lysate were diluted with 425 μl of buffer (K/Na—PO₄buffer, pH 7, 50 mM) and admixed with 25 μl of an approximately 220 mMsubstrate solution in DMSO (5 mg of protein/ml substrate concentrationapproximately 10 mM, 5% DMSO). The reaction was carried out in theThermomixer at 30° C. and 1000 rpm. After 0, 2, 4, 6, 8, 10, 15, 20, 30,60 and 120 minutes, in each case one Eppendorf was admixed with 0.5 mlof 1N HCl. After centrifugation (5 min, 13 000 rpm) and correspondingdilution, the concentrations of cyanohydrin and hydroxyamide weredetermined by HPLC. The activity of the nitrile hydratase was determinedfrom the velocity of formation of hydroxyamide (gradient in the initialrange). The standard substrate (100% activity) used was2-hydroxy-4-phenylbutyronitile. The activity in the hydrolysis of theother substrates was compared with the activity on2-hydroxy-4-phenylbutyronitrile (Tab. 5).

Results:

The activity of the nitrile hydratase in the crude lysate of the E. coliclone 7.3 in the hydrolysis of 2-hydroxy-4-phenylbutyronitrile wasapproximately 0.3 μmol·mg⁻¹·min⁻¹. In Table 5, activity on the differentsubstrates is compared. TABLE 5 Comparison of the activity of nitrilehydratase from E. coli clone 7.3 on the different substrates. Activityof the nitrile Substrate hydratase [%]

100

100

60Reaction of (R)-2-chloromandelonitrile on a Semipreparative Scale

50 ml of a crude lysate of the cloned nitrile hydratase from Rhodococcuserythropolis NCIMB 11540 (E. coli clone 7.3, produced according toExample 9) were diluted with 100 ml of buffer (K/Na—PO₄ buffer, 50 mM,pH 6.5). After adding 1.0 g of (R)-2-chloromandelonitrile (ee >99%) in1.5 ml of DMSO, the suspension was shaken at 150 rpm and 30° C. Aftercomplete reaction, the cell debris was centrifuged off and the productextracted continuously for 4 days with CH₂Cl₂.

ee of the crude product: >99%

Yield after purification: 0.91 g (82%)

1. A method for producing chiral α-hydroxycarboxylic acids, whichcomprises converting (R)- or (S)-cyanohydrins by enzymatic hydrolysis inthe presence of at least one suitable enzyme of a Rhodococcus speciesinto the conjugate (R)- or (S)-α-hydroxycarboxylic acids.
 2. The methodas claimed in claim 1, wherein the at least one suitable enzyme isselected from the group consisting of nitrilase and a combination ofnitrile hydratase and amidase.
 3. The method as claimed in claim 1,wherein the Rhodococcus species is selected from the group consisting ofRhodococcus erythropolis, Rhodococcus ruber and Rhodococcus rhodochrous.4. The method as claimed in claim 1, wherein the Rhodococcus species isRhodococcus erythropolis NCIMB 11540
 5. The method as claimed in claim1, characterized in that (R)- or (S)-cyanohydrins are used as startingmaterials which are obtained by enzymatic or chemically catalyzedaddition of a cyanide group to the corresponding aliphatic, aromatic orheteroaromatic aldehydes or ketones.
 6. The method as claimed in claim5, wherein the enzymatic addition of a cyanide group to thecorresponding aliphatic, aromatic or heteroatomic aldehydes or ketonesis catalyzed by a hydroxy nitrile lyase
 7. The method as claimed inclaim 6, wherein the hydroxy nitrile lyase is a hydroxy nitrile lyasefrom Hevea braziliensis or from Prunus anygdalis.
 8. The method asclaimed in claim 1, characterized in that (R)- or (S)-cyanohydrins ofthe formula

where R1 and R2 independently of one another are H, a C₁-C₆-alkyl orC₁-C₆-alkenyl radical which is optionally monosubstituted orpolysubstituted by substituents inert under the reaction conditions, ora phenyl radical which is optionally monosubstituted or polysubstitutedby substituents inert under the reaction conditions, with the provisothat R1 and R2 are not both H, are used.
 9. The method as claimed inclaim 1, characterized in that use is made of at least one suitableenzyme of a Rhodococcus species in the form of ground cells, crude orpurified enzymes, recombinant enzymes, immobilized cells or enzymes,lyophilized cells, or “resting cells”.
 10. The method as claimed inclaim 1, characterized in that the at least one suitable enzyme from aRhodococcus species is suspended in an aqueous medium and the resultantsuspension is admixed with the corresponding chiral cyanohydrin in thepresence of a solubilizer as cosolvent.
 11. The method as claimed inclaim 10, characterized in that, as solubilizer, use is made of organicsolvents, surfactants, or phase-transfer catalysts.
 12. The method asclaimed in claim 11, characterized in that, as organic solvent, use ismade of DMSO, DMF, C₁-C₆-alcohols, TMBE or mixtures thereof.
 13. Themethod as claimed in claim 10, characterized in that the cosolventfraction is between 0.5 and 20% by volume, based on the total volume ofthe reaction solution.
 14. The method as claimed in claim 1,characterized in that the pH of the reaction mixture is between 4.5 and11.
 15. The method as claimed in claim 1, characterized in that thehydrolysis is carried out at a temperature between 10 and 60° C.
 16. Themethod as claimed in claim 9, characterized in that, as recombinantenzyme, use is made of an enzyme obtained by expression of the pMS470plasmid system in a suitable host cell.
 17. A method for producingchiral α-hydroxycarboxylic acids which comprises converting (R)- or(S)-cyanohydrins by enzymatic hydrolysis in the presence of a nitrilasesinto the conjugate (R)- or (S)-α-hydroxycarboxylic acids and wherein the(R)- or (S)-cyanohydrines are obtained by hydroxy nitrile lyasecatalyzed addition of a cyanide group to the corresponding aliphatic,aromatic or heteroaromatic aldehydes or ketones.